Listeria monocytogenes is a facultative intracellular Gram-positive bacterium that causes invasive, often fatal, disease in susceptible hosts. As a foodborne pathogen, the bacterium has become a significant public health problem and has caused several epidemics in the United States and Europe. The virulence of L. monocytogenes is directly related to its ability to grow in the cytosol of host cells and its efficacy in spreading from cell to cell without leaving the intracellular milieu. We are interested in the mechanism regulating the activity of a bacterial phosphotipase C (PC-PLC), whose function increases the efficacy of bacterial cell-to-celt spread. PC-PLC is made as a proenzyme whose activation requires cleavage of a Nterminal prodomain. During intracellular growth, bacteria cumulate a pool of PC-PLC that is rapidly secreted in its active form upon a decrease in intracellular pH. A bacterial metalloprotease (Mpl) is involved in the regulation of PC-PLC activation and secretion. However, factors other than pH and Mpl control PC-PLC activation and secretion since a decrease in pH has little effect on the status of bacteria-associated PC-PLC in vitro. Our results reveal the existence of a previously undescribed mechanism regulating the activity of a virulence factor in a Gram-positive bacterial pathogen. This mechanism can be divided into three major steps: sorting, activation, and release of the virulence factor. Premature activation and secretion of PC-PLC in the cytosol of infected cells is cytotoxic, emphasizing the importance of this control mechanism for the intraceltular survival of L. monocytogenes. Our ultimate goal is to define at the cellular, molecular, and biochemical levels the mechanism regulating the activity of L. monocytogenes PC-PLC during infection. The specific aims of this proposal are to (I) Define functional regions of L. monocytogenes PC-PLC and Mpl that promote their association with the bacterial celt, (II) Identify and analyze bacterial factors accessory to the regulation of PC-PLC activity, and (III) Define environmental signals influencing the regulalation of PC-PLC activity. On a broader scope, these studies will contribute to our understanding of protein secretion in Grampositive bacteria.